SGLT1-based oral rehydration solution (ORS) used in the management of acute diarrhoea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements, isotope flux measurements in Ussing chambers, fluid movement in isolated ileal sacs and electrical measurements in patch clamp studies were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, SGLT1 and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestine and decreased whole-cell conductance in Caco-2 cells. In ileal intestinal sacs incubated with cholera toxin and glucose, decreased fluid secretion was seen only when the glucose-stimulated Cl secretion was blocked using niflumic acid. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. Glucose-induced chloride secretion may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhoea. The present results have immediate potential to improve the outcome in the treatment of acute and/or chronic diarrhoeal diseases.